An immobilized physiologically active substance obtained by immobilizing a physiologically active substance onto a carrier is used in various applications utilizing its biochemical reaction. Typical examples of such immobilized physiologically active substances are an immobilized enzyme obtained by immobilizing an enzyme onto a water-insoluble carrier, and a reagent for immuno diagnosis as obtained by immobilizing an immunologically active substance. The immobilzed enzyme has been put into practice in the industrial enzymatic reaction in recent years, and the reagent has been widely used in various types of diagnosis.
The enzymatic reaction is commercially employed in the process for production of medicines, foodstuffs and the like. In conventional methods, however, an enzyme is dissolved in an aqueous solution of a substrate and the enzymatic reaction is carried out in the resulting aqueous solution. In accordance with the above methods, however, it is very difficult to feed a fresh enzyme while maintaining predetermined reaction conditions, and to separate the reaction product and the enzyme without inactivation of the enzyme after the enzymatic reaction, and the enzyme is consumed uneconomically. Furthermore, productivity is low because the reaction is carried out batchwise. In order to overcome these problems, an immobilized enzyme as described above has been put into practical use. When a immobilized enzyme is used, the enzymatic reaction is carried out by contacting a substrate with the immobilized enzyme.
As a typical method for preparting such immobilized enzymes, a carrier bonding method is known, comprising bonding an enzyme to a water-insoluble carrier by covalent bonding, ion bonding or physical adsorption. Carriers which have heretofore been used are particles having a particle diameter of 1 mm to several millimeters, for example, derivatives of polysaccharides such as cellulose, dextran, agarose and the like, polyacrylamide, porous glass and the like. An immobilized enzyme obtained by using such particles is usually filled in a column, immobilized and contacted with a substrate solution. Therefore, if the substrate has a high molecular weight, problems arise in that the substrate is difficult to diffuse to the surface of the immobilized enzyme, a long period of time is required in the reaction, and the reaction conversion is low.
It has therefore been proposed to use latex particles as the carrier. For example, a typical latex conventionally used is produced by emulsion polymerizing a sparingly water-soluble radical polymerizable monomer such as styrene in the presence of an emulsifying agent and a water-soluble radical polymerization initiator.
The above emulsifying agent acts to secure polymerization stability during the process of emulsion polymerization and effectively permits to obtain polymer particles having a small particle diameter and a good dispersion stability. Although the reason why the emulsifying agent acts to increase dispersion stability of polymer particles is not necessarily clear, it is generally considered that part of the emulsifying agent is adsorbed onto polymer particles and the remainder is present in a free state in the medium, and that in an aqueous dispersion containing such water dispersion type polymer particles, an adsorption-desorption equilibrium exists between the emulsifying agent adsorbed onto the polymer particles and the free emulsifying agent and, as a result of the equilibrium, stabilization of dispersed polymer particles is attained.
When a latex containing an emulsifying agent is dispersed in a buffer or a physiological saline in order to immobilize a physiologically active substance such as an enzyme onto polymer particles, the above-described adsorption-desorption equilibrium of the emulsifying agent is lost and the dispersion stability of the polymer particles is deteriorated, resulting in aggregation and precipitation of the polymer particles, loss of the degree of freedom of the polymer particles and also a decrease in the biochemical reactivity. The free emulsifying agent often becomes an inhibitor for the biochemical reaction, e.g., the enzymatic reaction.
For this reason, in recent years, a method for preparing a latex by emulsion copolymerizing a monomer having an emulsifying ability by itself, such as sodium styrenesulfonate and polyethylene glycol monomethacrylate, and styrene in the absence of an emulsifying agent has been proposed. In the case of these latex particles, a physiologically active substance must be immobilized by the adsorption method because the latex particles do not have a functional group having a reactivity. This immobilization, however, produces various problems; for example, physiologically active substances which can be immobilized are limited, the pH range in which the immobilized physiologically active substance can be used is limited, and the storage stability is generally poor.
In addition, a method for immobilizing a physiologically active substance by covalently bonding the substance to a carboxylated latex such as a so-called carboxylated polystyrene obtained by copolymerizing styrene and methacrylic acid has been proposed. In general, however, the above conventionally known carboxylated latex has a poor dispersion stability and storage stability. In particular, when a large amount of a physiologically active substance is immobilized using the above latex particles or under the condition that a large amount of a physiologically active substance coexists with the latex particles, the latex particles easily coagulate and precipitate, leading to a serious reduction of the physiological activity. This tendency is marked particularly when the physiologically active substance-immobilized latex is dispersed in a buffer containing an organic solute or physiological saline.
The above-described immunological diagnosis reagent is a reagent for use in immunological diagnosis utilizing the immuno activity that a physiological active substance in the body liquid, such as blood, urine and other liquids to be tested, has. Such immunological diagnosis methods include a method in which an immuno active component is measured by utilizing a specific reaction occurring between an antigen or an antibody and the corresponding antibody or antigen when any one or both of the antigen and the antibody are reacted with a liquid to be tested, such as a body liquid, that is, an aggregation reaction or an aggregation inhibition reaction based on the antigen-antibody reaction. In this method, in order to facilitate the measurement with the naked eye or by an optical method, the antigen or antibody is usually deposited on a water-insoluble fine particle-shaped carrier, such as a latex and erythrocyte, to prepare a diagnosis reagent, and by utilizing the aggregation reaction of such particles, a component to be detected in the body liquid, such as serum, is measured.
The immunological diagnosis reagent is required to have a high sensitivity which, if only a small amount of an immunologically active substance is present in the liquid to be tested, permits to detect the immunologically active substance, and a high specificity which reacts only with the desired immuno active substance. Furthermore, the immunological diagnosis reagent is required to maintain its high detection sensitivity and specificity even when stored for a long period of time.
As such immunological diagnosis reagents, a diagnosis reagent in which polystyrene latex particles are used as the carrier and an antigen or antibody is immobilized on the surface of the particle by physical adsorption; a diagnosis reagent in which an antigen or antibody is immobilized by covalently bonding the antigen or antibody to carboxylated latex particles using carbodiimide, dialdehyde and the like; and so forth have heretofore been proposed. All of these reagents, however, have problems in that dispersion stability and storage stability are poor. Furthermore, when reacted with a liquid to be tested, those reagents sometimes cause the aggregation reaction not only with the corresponding positive substance but also with a negative substance. This aggregation reaction is called a "non-specific aggregation reaction". This non-specific aggregation reaction is a vital defect for the diagnosis reagent.
In recent years, in order to prevent the above non-specific aggregation reaction of latex, there has been developed the latex immuno nephelometry in which a liquid to be detected, such as serum and urine, is diluted to several hundred times to several thousand times the original volume thereof and reacted with a latex in an optical cell, and the latex aggregation reaction based on the antigen-antibody reaction is determined by an absorbance of visible light, near infrared light, ultraviolet light, laser light and the like, or by an optical change in turbidity, scattering and the like.
In this method, however, the dilution of the liquid to be detected to a great extent as described above naturally causes a marked reduction in sensitivity and further increases a deviation in optical change. Therefore, this method is poor in reproductivity of measurement. On the other hand, if the amount of the antigen or antibody, for example, immobilized on the latex is increased in order to increase the sensitivity of the latex reagent, the deviation in optical change is increased and the concentration of antigen or antibody in the liquid to be detected which can be measured is limited. Furthermore, as described above, the non-specific aggregation reaction rather readily occurs.